Cereal crops are very important to a majority of the world's populations. For instance, wheat is the most important staple food of about two billion people (36% of the world population). Worldwide, wheat provides nearly 55% of the carbohydrates and 20% of the food calories consumed globally. It exceeds in acreage and production every other grain crop (including rice, maize, etc.) and is cultivated over a wide range of climatic conditions and the understanding of genetics and genome organization using molecular markers is of great value for genetic and plant breeding purposes.
The world's main wheat producing regions are China, India, United States, Russian Federation, France, Australia, Germany, Ukraine, Canada, Turkey, Pakistan, Argentina, Kazakhstan and United Kingdom. Most of the currently cultivated wheat varieties belong to Triticum aestivum L., which is known as common bread wheat and valued for bread making. The greatest portion of the wheat flour produced is used for bread making.
Bread wheat is a hexaploid, with three complete genomes termed A, B and D in the nucleus of each cell. Each of these genomes is almost twice the size of the human genome and consists of around 5,500 million nucleotides. On the other hand, durum wheat, also known as macaroni wheat or pasta wheat (Triticum durum or Triticum turgidum subsp. durum), is the major tetraploid species of wheat of commercial importance, which is widely cultivated today. Durum wheat has two complete genomes, termed the A and B genomes.
Wheat is a widely studied plant, but in some cases, development of new traits is hampered by limited genetic diversity in today's commercial wheat cultivars and also because the bread wheat genome typically has three functionally redundant copies of each gene (called homoeologs), and therefore, single gene alterations usually do not produce any readily visible phenotype such as those that have been found in diploid corn. Often in bread wheat, altered variants of all three homoeologs must be combined genetically in order to evaluate their effects.
Whole grain products present a challenge to the wheat industry because whole grain flour has a greatly reduced shelf life compared to refined flour (Doblado-Maldonado 2012). The reduced shelf life of whole grain flour is due primarily to degradation of lipids, which are the most unstable components of the milled whole grain (Pomeranz 1988; Tait and Gailliard 1998). Lipid degradation leads to the production of bitter, rancid and off flavors that negatively impact the shelf life and use of whole grain flour and products made from it. Similarly, stability and reduced shelf life are issues for the use of rice bran and products derived from it such as rice bran oil, due to the degradation of lipids.
The degradation of lipids occurs through the processes of both hydrolytic and oxidative rancidity. Lipases (EC 3.1.1.3) catalyze the hydrolysis of ester bonds in mono-, di- and tri-acylglycerides (TAG) into non-esterified or free fatty acids (FFA). Lipid degradation begins immediately upon milling whole grain flour or rice bran. Lipases have substantial enzymatic activity even at the low moisture content of milled grain (typically 10-14% for wheat) (Doblado-Maldonado 2012). Lipids can also be further degraded through autooxidation or by lipoxygenases (Lpx's). Lpx's (EC 1.13.11.12) are a class of non-heme iron-containing dioxygenases that catalyse the positional and specific dioxygenation of polyunsaturated fatty acids that contain 1,4-cis,cis pentadiene structures to produce the corresponding hydroperoxides. Following the formation of hydroperoxides, further degradation of lipids leads to the formation of smaller volatile compounds such as epoxyaldehydes, ketones, furans and lactones that cause off flavors and odors and reduce the shelf life of materials. Attempts to inactivate or reduce lipase and lipoxygenase activity in whole grain flour or rice bran by microwave, heat, vacuum, cold storage or chemical treatment have met with limited success or are very expensive to employ commercially.
Relatively few plant lipases have been characterized at the molecular and biochemical level (Seth et al. 2014 Protein Exp and Pur 95:13-21). For example, the rice genome annotation project currently has 73 genes annotated as putative lipases (Kawahara et al. (2013) Rice 6:4), but difficulties encountered in purification of rice lipase enzymes has hindered their characterization (Muniandy, K., et al. The Nucleus (2015): 1-6). Despite these difficulties, several rice bran lipolytic enzymes have been identified biochemically including Lipase I, Lipase II, a thermally stable lipase and an esterase (OsEST-b), (reviewed in Muniandy 2015 and Chuang et al. Journal of agricultural and food chemistry 59.5 (2011): 2019-2025). Of these enzymes, Lipase II and OsEST-b were recently purified and the rice genes coding for the enzymes have been identified (Vijayakumar and Protein expression and purification 88.1 (2013): 67-79; Chuang et al. 2011).
In wheat, lipase activities have been described in multiple tissues, including ungerminated wheat bran and wheat germ fractions as well as lipases produced during germination (Pomeranz, Y. Wheat: chemistry and technology. No. Ed. 3. American Association of Cereal Chemists, 1988). However, the genes coding for these lipases are largely uncharacterized. The present inventors have identified the Lipase 1 genes in wheat, in which the expression is located in the grain of the wheat plant.
The inventors have identified novel human induced non-transgenic Lip1 mutations and analyzed their phenotypes in plants, such as wheat and rice. With regard to wheat, since multiple Lipase genes (Lip1, 2 and 3) are all expressed, each with one or more potential homoeologs in the A, B, and D genomes, it is unclear if altering one gene or gene family could positively affect shelf life or stability of whole grain flour in bread wheat. Mutations in the Lip1 genes in the wheat genome or rice genome provide a potential pathway for providing increased hydrolytic and oxidative stability in wheat/wheat flour and rice bran and products derived therefrom. The disclosure herein demonstrates that novel alleles in the Lip1 gene significantly improve shelf-life.